In the life science field, a transcription activity of an intracellular gene has been generally determined, and used for evaluation of exogenous factors given to cells, and analyses of intracellular signal transduction or expression of an individual protein group. The gene transcription activity has been directly determined by Western blotting and the like, or indirectly determined using a luciferase gene or a light-emitting enzyme gene as a reporter gene. In particular, it has been generalized to quantify the transcription activity based on an emitted light intensity using a firefly light-emitting enzyme gene. A fluorescent protein exhibits a fluorescent activity without need of a cofactor almost simultaneously with its intracellular expression. The fluorescent protein has been used as a monitor protein for examining a localization of a protein by the use of the fluorescent activity in the cell as an indicator, but it is difficult to quantify it, and it is unlikely to use it as the reporter gene for the gene expression.
It is important to analyze a quantitative and temporal dynamic change of the protein gene expression, but the transcription activity one gene has been primarily analyzed in conventional reporter techniques. However recently, a system (dual assay system, Promega) for determining two transcription activities by introducing two gene constructs into the cell, i.e., A transcription active region being inserted in a firefly light-emitting enzyme gene and B transcription active region being inserted in a Renilla light-emitting enzyme gene has been commercially available. However, this method is a system for determining the transcription activity by adding different luminescent substrates, respectively, two activities can not be determined simultaneously, and only two transcription activities can be determined. Furthermore, since a firefly luciferase is used, a wavelength thereof is changed due to pH and accurate determination is difficult.
Multiple signals are trafficked in a cell, and it is essential to construct a technique to quantitatively determine the multiple transcription activities. For example, in a human biological clock, a Per gene which gives a 24 hour rhythm is controlled by Clock and BMAL gene products. Thus, to precisely evaluate the biological clock, it is essential to determine multiple, at least three transcription activities. Until now, the transcription activity of an individual gene has been determined by the use of a firefly luciferase reporter gene, but a dynamic of only one gene transcription has been observed at a time, and an interaction of biological clock-related gene expressions has remained unclear.
Canceration progresses by abnormal growth of cells caused with activation of an oncogene or by the abnormal growth of the cells due to the control release caused along with inactivation of a tumor suppressing gene. Thus, to evaluate canceration factors and intracellular signal transduction of the canceration, it is desirable to determine the gene transcription activity of the oncogene, the tumor suppressing gene and a mitotic marker gene. However, in the conventional method, the dynamic of only one gene transcription has been observed at a time, the transcription activities of the three gene can not be evaluated at a time, and thus the interaction of the three genes involved in the canceration has not been sufficiently understood.
The transcription of a gene is caused by binding a substance which suppresses or promotes the gene expression to a particular sequence present on a gene sequence referred to as a promoter region upstream of a gene product. An E-box and a cAMP-binding site are representatives thereof. The gene transcription activity is determined by inserting a certain length of the promoter region into an upstream of a reporter gene. Furthermore, a particular sequence believed to be effective is then synthesized, and inserted into the upstream of the reporter gene to examine an effect of the particular sequence. To examine a transcription controlling effect of the particular sequence, it is necessary to simultaneously evaluate the transcription activity of the original promoter region and the transcription activity capable of standardizing the effect in combination. However, in the conventional method, the transcription dynamic of only one gene has been observed, and the particular sequence for the control of the transcription activity can not be sufficiently evaluated.
A luciferase is useful as a means to directly observe the gene transcription activity in the cells, and has been used as a detection monitor protein of the gene expression. There are a wide variety of luciferases, but no reporter gene for determining the transcription activity based on their diversity is available. If using luciferase genes which emit different color lights as the reporter genes and different transcriptional active regions are inserted into mammalian cells, then multiple transcription activities can be determined. A red-emitting luciferase derived from a rail road worm has the longest wavelength of luminescence, is easily discriminated compared to the luciferases derived from a firefly and a click beetle, and is highly permeable into the cell due to the red-emitting color. However, the expression of the red and green-emitting luciferases from the rail road worm has been successfully done only in Escherichia coli (US 2002/0119542-A1), and there is no successful example as the system in the mammalian cells including human cells.
There is also an example in which the expression of a luciferase gene from the rail road worm in the mammalian cells was enabled by modifying a structure of the gene (WO 2003/016839).
As the luciferase, a luciferase derived from Rhagophthalmus ohba has been also known.
The expression of a green-emitting luciferase derived from Rhagophthalmus ohba has been successfully done in only Escherichia coli (Ohmiya, Y. Sumiya, M. Viviani, V R. and Ohba N.; Comparative aspects of a luciferase molecule from the Japanese luminous beetle Rhagophthalmus ohba. Sci. Rept. Yokosuka City Mus. .47, 31-38, 2000). Based on this sequence, an orange-emitting luciferase derived from Rhagophthalmus ohba was created and the expression thereof was also successfully done in Escherichia coli (Viviani, V R., Uchida, A., Suenaga, N., Ryufuku M. and Ohmiya Y.: Thr-226 is a key-residue for bioluminescence spectra determination in beetle luciferases. Biochem. Biophys. Res. Commun., 280, 1286-1291, 2001). Additionally, as the luciferase, blue-emitting luciferases derived from a dinoflagellate and Renilla have been also known.
It is an object of the present invention to construct and optimize a reporter gene capable of determining or quantifying multiple transcription activities in a cell simultaneously or at the same phase, further develop a multiple gene transcription activity determining system using the reporter gene group, and utilize the same for cell functional analyses in life science, further the treatment/examination of pathology and new drug development.
It is also another object to make a gene construct by which a red- or a green-emitting luciferase from a rail road worm is stably transcribed and stably translated in mammalian cells or in animals.
It is also another object to make a gene construct by which an orange or a green-emitting luciferase from a Rhagophthalmus ohba is stably transcribed and stably translated in mammalian cells or in animals.
This enables to stably determine and visualize a change of the gene transcription activity in the mammalian cells or in the animals.